Abstract: As an effective tool for the research on gene discovery and gene expression profile, expressed sequence tags (EST) have been taken into account by many laboratories in the world. Reducing the cost of large-scale EST sequencing and improving the success rate of EST sequencing are particularly important for relevant research. Based on different comparison between such factors as response System,BigDye type, content of BigDye,purification of sequence product and 96-well reaction plate,the relative cheap project of large-scale EST sequencing has been confirmed. The quality of cDNA library and other influence factors have been discussed at the same time. It is significant and useful for reducing the cost and improving the success rate of EST sequencing.